bioRxiv preprint doi: . this ...60 collectively defined as “xero-protectants” (Loi et al.,...

Late Embryogenesis Abundant (LEA) proteins confer water stress tolerance to 1

mammalian somatic cells 2

3

Czernik M1;2;7., Fidanza A1;3;7., Luongo FP1;4., Valbonetti L5., Scapolo PA1., Patrizio P6., 4

Loi P1* 5

6

1 Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy 7

2 Department of Experimental Embryology, Institute of Genetics and Animal Breeding, Polish 8

Academy of Sciences, Jastrzebiec, Poland 9

3 Current address: Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK 10

4 Current address: Center for Neurovirology, Department of Neuroscience, Lewis Katz School 11

of Medicine at Temple University, Philadelphia, PA 19140, USA 12

5 Faculty of Biosciences and Technology for Food, Agriculture and Environment, University of 13

Teramo, 64100 Teramo, Italy. 14

6 Yale Fertility Center, New Haven, CT 06511, USA. 15

7 co-first authorship 16

* Corresponding author 17

18

Corresponding author: Pasqualino Loi, Faculty of Veterinary Medicine, University of 19

Teramo, 64100 Teramo, Italy; phone number: +39 0861 266 856; [email protected], 20

21

22

Key words: Late Embryogenesis Abundant (LEA) proteins, xeroprotectants, mammalian 23

somatic cells, desiccation, 24

25

26

.CC-BY-NC-ND 4.0 International licensecertified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which was notthis version posted July 16, 2019. . https://doi.org/10.1101/704809doi: bioRxiv preprint

https://doi.org/10.1101/704809

http://creativecommons.org/licenses/by-nc-nd/4.0/

Abstract 27

Late Embryogenesis Abundant (LEA) proteins are commonly found in organisms capable 28

of undergoing reversible dehydration - “anhydrobiosis”. Here, we have produced three 29

LEA proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-30

cytoplasm; pTag-WCOR410-RFP, Tricum aestivum cold acclimation protein WCOR410, 31

binding to cellular membranes, and pTag-LEA-BFP, Artemia franciscana LEA protein 32

group 3 that targets the mitochondria. Somatic cells transfected with three LEA proteins 33

were subjected to desiccation under controlled conditions, followed by rehydration, 34

viability assessment and membrane/mitochondria functional tests were performed. Results 35

shown that LEA protect cells from desiccation injury. Cells expressed all LEA proteins 36

shown very high percentage of viable cells (58%) after four hour of desiccation compare to 37

un-transfected cells (1% cell alive). Plasmalemma, cytoskeleton and mitochondria 38

appeared unaffected in LEA-expressing cells, confirming their protective action during the 39

entire desiccation and rehydration process. Here, we show that natural xeroprotectants 40

(LEA proteins) transiently expressed in somatic cells confer them desiccation tolerance. 41

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Introduction 53

Water is essential for life (Hand et al., 2007; Menze et al., 2009) yet many organisms are 54

able to survive almost completely dehydrated (>99% of their body water is removed) 55

(Crow et al., 1992). In nature, this phenomenon is known as “anhydrobiosis”, and is 56

conserved across vegetal and animal phyla (Hincha et al., 1992). Anhydrobiosis allows 57

seeds and small invertebrates to survive long time spans (decades/centuries) in the absence 58

of water, thanks to the induced synthesis of sugars and various protein classes that can be 59

collectively defined as “xero-protectants” (Loi et al., 2013). Among them, Late 60

Embryogenesis Abundant proteins (LEAp) are the best characterized and perhaps the most 61

interesting (Marunde et al., 2013). LEA proteins were first discovered in cotton seeds more 62

than 30 years ago (Dure et al., 1981) and were later also found in seeds and vegetative 63

tissues of several other plants (Shih et al., 2008). A relatively recent survey, probably not 64

updated, contains 769 LEAp entries from 196 organisms (Hunault and Jaspard, 2009). 65

LEA proteins are highly hydrophilic and acquire random coils conformation in aqueous 66

solution, property that has assigned them the definition of “intrinsically disordered” 67

proteins (McCubbin et al., 1985). It is only during de-hydration that LEA proteins acquire 68

their final conformations, primarily α helices, β sheets, and hairpin loops, and by doing so 69

they bind to specific cellular/enzymatic substrates to be protected. The mechanism of 70

“xero-protection” is not fully understood. Also lacking are data on the substrate binding 71

mechanism. Data gained in model organisms have indicated that LEAp stabilisation occurs 72

via several pathways: chaperon-like activity, protection of cell membranes, stabilisation of 73

vitrified sugar glasses by increasing glass transition temperature (Tg), sequestration of 74

divalent ions, and synergic interaction with other xero-protectants, such as trehalose (Li et 75

al., 2012). The expression of LEAp, as well as the other xero-protectants, is triggered in 76

anhydrobiotic organisms once water stress is sensed, leading to a progressive accumulation 77

in various cellular compartments, such as mitochondria (Hand et al., 2011; Moore and 78


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Hand, 2016), nucleus (Wu et al., 2013) cytosol (hand et al., 2011), membranes (Tolleter et 79

al., 2010), and endoplasmic reticulum. Clearly, several LEAp are required, along with 80

other xero-protectants, to confer full protection from water stress. Therefore, if the strategy 81

is to exploit LEAp for the induction of reversible drying in mammalian cells, it is 82

necessary to know their specific targets and the mechanism of action. While the 83

mechanism of action might be inferred from sequence analysis of the LEAp by 84

bioinformatics, in vivo transfection assays of cells with the different LEAp are required to 85

confirm their protective action, but also to exclude negative effects on cell homeostasis. 86

After all, they are mainly vegetal proteins. An index paper published by Li’s group was the 87

first to investigate the effects of LEAp expressed in mammalian cells subjected to rapid 88

dehydration (Li et al., 2012). In that work, hepatoma cell line was stably transfected with a 89

tetracycline (Tet)-inducible expression system coding for two LEAp naturally expressed in 90

embryos of the brine shrimp Artemia franciscana, AfrLEA2 and AfrLEA3m, plus a 91

trehalose transporter 1 (TRET1) (Li et al., 2012). The results showed that LEAp, one 92

expressed in the cytoplasm, AfrLEA2, and the other AfrLEA3m in the mitochondria, 93

together with trehalose, effectively protected the cells from the desiccation stress. 94

Our work extends Li’s findings. In addition to the LEAp AfrLEA3m, that targets the 95

mitochondria, we have transfected primary cultures of fibroblasts with two additional LEA 96

proteins: pTag-RAB17-GFP-N, Zea mays dehydrin-1dhn, expressed in the nucleo-97

cytoplasm; and pTag- WCOR410-RFP, Tricum aestivum cold acclimation protein 98

WCOR410, that binds to the membranes. Our preliminary work on lyophilized, 99

unprotected, somatic cells directly processed for scanning and electron microscopy showed 100

massive damage at the membrane level [(Matzukawa, personal communication and (Iuso et 101

al., 2012)]. Here reported results show that the individual LEAp protected the cells from 102

desiccation with strongest effect when all three LEA were expressed adding new data on 103

the induction of controlled drying in mammalian cells. 104


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Results 105

Subcellular localization of pTag-RAB17-GFP, pTag-WCOR410-RFP, and pTag-106

LEA-BFP 107

Sheep fibroblasts were transiently transfected with pTag-RAB17-GFP, pTag-WCOR410-108

RFP, pTag-LEA-BFP with efficiency as follow: 44%, 26% and 24%, respectively. pTag-109

RAB17-GFP was localised to the cytoplasm and nucleus (Fig. 1A-C), pTag-WCOR410-110

RFP protein was observed only in the cytosol/membranes (Fig. 1G-S) and by localization 111

with membrane dye clearly shown proper localization (Fig. 1M – S), while pTag-LEA-112

BFP was detected in the mitochondria (Fig.1T - Z). Fig. 1W-Z shows that pTag-LEA-BFP 113

is targeted to the mitochondrial network as it co-localised with MitoTracker green (Fig. 114

1X, Z). Additionally, to confirm the proper localization of the LEA proteins, sheep 115

fibroblasts were transfected with empty vectors EV-GFP, EV-RFP and EV-BFP as a 116

control. Results showed that GFP, RFP and BFP alone had spread distribution throughout 117

the cells (Fig. 1D-F: GFP; Fig. 1J-L: RFP; Fig. 1U: BFP). Expression of fusion LEA 118

proteins in sheep fibroblast was also confirmed by immunoblotting analysis (Fig. 1Z’). 119

Moreover, to our knowledge for the first time, we were able to express all tree LEA 120

proteins (pTag-WCOR410-RFP (Fig. 2A); pTag-RAB17-GFP (Fig. 2B); pTag-LEA-BFP 121

(Fig. 2C)) in the same somatic cell, although with low efficiency (11%). Additionally, in 122

the MIX conditions we have also found cells that expressed only two LEA protein (12%) 123

as well as cells with single LEAp: pTag-RAB17-GFP (21%), pTag-WCOR410-RFP (29 124

%), or pTag-LEA-BFP (14%). 125

126

Enhanced resistance to desiccation stress 127

Sheep fibroblast transfected with LEA proteins, as well as not transfected controls (CTR), 128

were air dried at 16°C for up to 4h. Every 60 min, cells viability was assessed on sub-129

samples using trypan blue exclusion. The results showed that 1h of air drying did not affect 130


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viability of sheep fibroblasts (Fig. 3A), with more than 80% of cells expressing any of the 131

LEAp singularly or together and 60% of CTR maintaining viability (Fig. 3A). One hour 132

later small negative effect of desiccation on cells viability start to be observed but any 133

statistic differences between LEAs and CTR groups were observed. Drastic differences 134

were observed 1h later (3h after initiation of desiccation). The numbers of viable cells 135

expressing a single LEA protein were significantly higher than control group (RAB-17: 136

16%, WCORB410: 13%, LEA3: 12%, CTR: 2%). By 4h after desiccation begun, only few 137

sheep fibroblast in the CTR were still alive (less than 1 %) while LEA proteins were able 138

to protect the somatic cells, as indicated by their viability (Rab17: 8%, WCOR410: 5%, 139

LEA3: 2.3%) (Fig. 3A), with the stronger effect observed when all three LEA proteins 140

were co-transfected together (LEA-MIX). In this group, 40% of LEA-MIX transfected 141

cells were still alive after 3h of air drying, compared to 2% of the CTR group; 23% of MIX 142

cells were viable after 4h of drying while in the control group viability dropped to under 143

1% (Fig. 3A). 144

To verify viability every hour, we were forced to remove the cells from the drying 145

chamber, and by doing so we exposed them to uncontrolled variation in both humidity and 146

temperature. This could have affected the late time points. For this reason, we decided to 147

carry on the viability tests at two time points: 1h and 4hs post desiccation. Accordingly, 148

stronger difference in cells viability was observed when cells were not exposed to 149

condition changes. Indeed, after 4h of desiccation, statistically significant difference 150

between cells expressing single as well as all three LEA together (MIX) and the control 151

group were observed (RAB17: 40%, WCOR410: 34%, LEA: 37%, MIX: 58%, CTR: 2%) 152

(Fig. 3B). 153

154

LEA proteins preserve proliferation capacity following desiccation 155


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Transfection of a single LEA protein resulted in a higher number of cells attached to 156

culture dishes (pTag-RAB17-GFP: 55 cells/field; pTag-WCOR410-RFP 62 cells/field; 157

pTag-LEA-BFP: 37 cells/field; LEA-MIX: 86 cells/field) than in cells desiccated without 158

LEA, CTR-D (non- transfected and desiccated cells) (25 cells/field), (Fig.4B). Proliferation 159

rate was 32% with pTag-RAB17-GFP; 31% with pTag-WCOR410-RFP; and 25% with 160

pTag-LEA-BFP (Fig. 4A). In the LEA-MIX group, cell proliferation rate was at levels 161

comparable to the non-desiccated controls (CTR) (48% vs 51%, respectively). 162

163

Cellular integrity of mammalian cells after desiccation 164

Somatic cells transfected with LEA proteins and controls (non- transfected and desiccated 165

– CTR-D), were cultured for an additional 24h after desiccation and rehydration. Non-166

transfected and non-desiccated cells were used as a positive control (CTR). Results showed 167

that LEA proteins protected cellular organelles from desiccation injury (Fig. 5). Cells 168

expressing pTag-WCOR410-RFP (Fig. 5A-H) and pTag-RAB17-GFP (Fig. 5I-R) did not 169

show any post-desiccation damages of the cytoskeleton as shown by F-actin staining (Fig. 170

5A, O). Normal actin filaments, spanning the entire cells, were observed in sheep 171

fibroblast expressing these LEA proteins, compared to the CTR group (Fig. 5T). CTR-D 172

cells were less organised and showed fragmented cytoskeleton (Fig. 5Y). Importantly, 173

LEA proteins, and particularly pTag-LEA-BFP, protected the mitochondria in transfected 174

cells (Fig. 6A-D), where high numbers of active mitochondria localized in perinuclear 175

position (Fig. 6A), were observed, similar to the CTR group (Fig. 6E-H). On the other 176

hand, mitochondrial activity was very poor in CTR-D group (Fig. 6I-L) where the 177

organelles were localized peripherally, displaying high fragmentation, and were much less 178

metabolically (Fig. 6I). 179

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Discussion 182

The natural capacity of simple organisms to survive in a dehydrated state has long been 183

exploited by humankind, with lyophilization as the method of choice for the long-term 184

storage of bacteria and yeast (Fonseca et al., 2015). Some attempts were subsequently 185

conducted to freeze dry non-nucleated mammalian cells, like platelets and red blood cells, 186

with partial success (Crowe et al., 2003). It was the report of the maintenance of nuclear 187

viability in lyophilized spermatozoa (Wakayama and Yanagimachi, 1998) that sparked the 188

interest in dry stabilization of mammalian cells. The low water content and the highly 189

condensed DNA make spermatozoa “easy” to dry, while in contrast other mammalian cells 190

are not tolerant to dehydration and invariably die. No genes related to LEA family have 191

been identified in the mammalian genomes sequences so far, beside one short protein 192

conferring mitochondrial protection (Hall et al., 2011). Therefore, the only possible way to 193

confer cell desiccation tolerance is to provide them with suitable xero-protectants. 194

195

The first studies reporting on the possibility of drying somatic cells were published by Guo 196

et al., (2000) and Eroglu et al., (2000). These authors showed that it was possible to 197

desiccate and store human fibroblasts for up to 5 days while maintaining viability upon re-198

hydration. Their method made use of the protective effects of trehalose, a disaccharide 199

associated with organisms withstanding desiccation (Leslie et al., 1995; Welsh and 200

Herbert, 1999). Trehalose was produced in cells previously infected with an adenoviral 201

vector expressing the trehalose biosynthetic genes, otsA and otsB, followed by air-drying 202

and storage at room temperature. 203

Here we followed this general strategy, but LEA proteins were used as xero-protectants. 204

Our work builds on a recent paper where desiccation tolerance was induced in hepatoma 205

cell line expressing Tet-inducible expression system coding for two LEA proteins of the 206

brine shrimp Artemia franciscana, AfrLEA2 and AfrLEA3m, and a trehalose transporter 1 207


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(TRET1). Of the two LEA proteins, AfrLEA2 accumulated in the cytoplasm, and 208

AfrLEA3m selectively targeted the mitochondria. Here we maintained the latter LEA 209

protein, but we selected two different additional ones. In our previous work on 210

lyophilization and nuclear transfer of lyophilized cells, we found high level of DNA 211

damage in the resulting pronuclei (Iuso et al., 2012). Therefore, we elected to use pTag-212

RAB17-GFP-N, Zea mays dehydrin-1dhn, that is expressed not only in the cytoplasm as 213

AfrLEA2 does, but also in the nucleoplasm, to protect the DNA as well. The third LEA 214

protein that we utilized was pTag-WCOR410-RFP Tricum aestivum cold acclimation 215

protein WCOR410, that binds specifically to the membranes. This latest LEA proteins was 216

included following our observation of a massive membrane damage in lyophilized 217

fibroblasts processed for scanning electron microscopy (SEM) without re-hydration 218

(Matsukawa, unpublished) and for transmission electron microscopy (TEM) after re-219

hydration (Iuso et al., 2012). 220

221

The proportion of fibroblasts expressing single LEA proteins was around 30%, with no 222

significant differences between the three vectors. Transfection efficiency of all three LEA 223

expression vectors was much lower, about 11%. As a result, the data on cell survival were 224

underestimated because desiccated samples contained both expressing and non-expressing 225

cells. Transgenic cell lines constitutively expressing xero-protectants, as accomplished in 226

Li and co-workers (Li et al., 2012), provide indeed more precise and objective findings, but 227

such approach cannot be an option for practical use. 228

The subcellular localization of all three LEA proteins matched the expectations (Fig. 1 and 229

2), confirming an earlier report (Li et al., 2012). No adverse effects on cell viability were 230

observed in the LEAp expressing cells. 231

The LEA proteins exerted protection against water deprivation, with no major differences 232

between them. LEA pTag-RAB17 expression appeared to be more beneficial over the 233


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other two, probably owing to its ubiquitous expression in all cell compartments (Fig. 3 and 234

4), and also because of its higher transfection efficiency compared to the other two [(44% 235

vs 26% (WCOR410-RFP) and 24% (LEA3-BFP)]. Clearly, cells expressing all three LEA 236

proteins showed the best survival rate, particularly in the experiment with only two time 237

point controls (Fig. 3B). Expression rate of all LEA proteins in the same cells was low 238

(11%) but presence of other LEAp combinations (single LEAp expression, and 239

combinations of two LEAp in the same cell) increased the protective effects in the MIX 240

group. 241

The proliferation assays after the water stress further demonstrated the beneficial effects of 242

LEA proteins, basically in the cell functions explored - mitochondrial function and 243

distribution, and F-actin (cytoskeleton). Again, no major differences between cells 244

expressing individual LEA protein were detected (Fig. 5), while those expressing the three 245

LEA proteins showed remarkable growth performances, comparable to control, unstressed 246

cells (CTR) (Fig. 4A). Viability was further supported by the normal number and 247

distribution of the mitochondria in growing cells, as well as the normal organization of 248

polarized F-actin across the cells (Fig. 5). In contrary, unprotected cells displayed 249

abnormal mitochondrial distribution (Fig. 6I) and disordered F-actin scattering across the 250

cells (Fig. 5Y), suggesting that time is needed to recover from desiccation damages. 251

252

As learned from anhydrobiotic models, desiccation tolerance is conferred through a 253

combined action of several xero-protectants, including LEA proteins. Our work using a 254

peculiar combination of three LEAp expressing plasmid vectors transferred into sheep 255

fibroblasts, reconfirmed their predicted localizations (Fig. 1), and convincingly 256

demonstrated their protective effects during dehydration, the recovery on rehydration and 257

continued growth following in vitro culture. Our data ameliorate our knowledge on the 258


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induction of reversible drying in mammalian nucleated cells for their long-term 259

stabilization in an anhydrous state as an alternative biobanking approach. 260

261

Materials and methods 262

Construction of LEAp plasmids 263

Coding Sequences (CDS) for the LEA protein were produced by gene synthesis (Dundee 264

Cell Products, USA): RAB17 (~0.5 Kb; Zea mays dehydrin-1dhn, GenBank 265

NM_001111949.1), WCOR410 (~0.8 Kb; Tricum aestivum cold acclimation protein 266

WCOR410; GenBank L29152.1) and LEA (~0.9 Kb; Artemia franciscana LEAp group 3; 267

GenBank FJ592175.1). RAB17, WCOR410 and LEA were subcloned into pET-15b 268

(Novagen, Rome, Italy) under T7 promoter. Subsequently, CDS were amplified using 269

AccuPrime Pfx DNA polymerase (ThermoFisher), and inserted using EcoRI/HindIII into 270

the pTag-GFP-N, pTag-RFP-N to obtain pRAB17-GFP and pWCOR410-RFP, and 271

SacI/PstI for the pTag-BFP-N to obtain pLEA-BFP (all plasmid backbones were from 272

Evrogen, Milano, Italy). Correct clones were confirmed by Sanger sequencing using ABI 273

PRISM 3100 (Applied Biosystem). 274

275

Transfection 276

Sheep adult fibroblast (SAF) were derived from ear biopsy of three female Sarda breed 277

sheep (2 years old). Animal work (skin biopsy) has been approved by the Italian Ministry 278

of Health, upon the presentation of the research description prepared by the ethics 279

committee of the Istituto Zooprofilattico Sperimentale di Teramo (Prot. 944F0.1 del 280

04/11/2016). The number of the authorization granted by the Italian Ministry of Health is 281

n° 200/2017-PR. We confirm that all methods were performed in accordance with the 282

relevant guidelines and regulations. 283

284


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SAFs (between second and eighth passage) were cultured in DMEM (GIBCO) containing 285

2 nM glutamine, 3.7 g/L NaHCO3 and 0.5% gentamicin supplemented with 10% Fetal 286

Bovine Serum (FBS). 287

Transfection of sheep adult fibroblasts was adapted from Czernik and colleagues (Czernik 288

et al., 2016) with small modifications. Approximately 106 cells were plated in 3,56 cm 289

dishes and cultured in Minimal Essential Medium (MEM) + 10% foetal bovina serum 290

(FBS) (Gibco, Milan, Italy) for 24h. After 24h cells were transfected using Lipofectamine 291

2000 kit (Invitrogen, Milan Italy), according to the manufacturer’s protocol, using 3μg of 292

pTag-RAB17-GFP-N, pTag-WCOR410-RFP-N, pTag-LEA-BFP, -individually or in 293

combination (by 2μg of each) (called MIX). Additionally, empty vectors: pTags-GFP-N, 294

pTags-RFP-N, pTags-BFP-N were used as a control (called EV-GFP, EV-RFP, EV-BFP, 295

respectively). After transfection cells were incubated in a humidified atmosphere 5% 296

CO2/95% air at 37°C. All experiments were done 24h post-transfection. Efficiency of the 297

transfection of all experiments were normalised normalized by using an internal control, 298

299

Localization of LEA proteins in somatic cells 300

Sheep fibroblasts transfected with pTag-RAB17-GFP, pTag-WCOR410-RFP, pTag-LEA-301

BFP individually or in combination, as well as empty vector controls, EV-GFP, EV-RFP, 302

EV-BFP, were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature 303

(RT). After subsequent wash with PBS, cells were counterstained for 10min with 5µg/mL 304

Hoechst 33342 (pTag-RAB17-GFP and pTag-WCOR410-RFP cells) or 0.5μg/mL 305

Propidium Iodide (PI) (pTag-LEA-BFP expressed cells). Then, cells were mounted on 306

slides with Fluoromount™ aqueous mounting medium (Sigma, Milan, Italy) and 307

localization of the proteins was analysed with using Nikon Ar1 laser confocal scanning 308

microscope (Nikon Eclipse Ti-E) equipped with the NIS- Element 4.40 software. 309

310


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Western Blot 311

Proteins were isolated from sheep fibroblasts transfected with pTag-RAB17-GFP, pTag-312

WCOR410-RFP, pTag-LEA-BFP, EV-GFP, EV-RFP, EV-BFP and from the mock 313

transfection control, by incubation overnight with lysis buffer I (20 mM Tris, 150 Mm 314

NaCl2, 1% NP-40) at 4°C. Then protein extracts were re-suspended at 1:1 ratio in lysis 315

buffer II (20 Mm Tris, 150 Mm NaCl2). Protein concentration was assessed using the BCA 316

Protein Assay Kit (Thermo-Fisher, Milan, Italy) according to the manufacture protocol. 317

For each sample 50 µg of protein were incubated at 95°C for 10 min and then loaded into a 318

gradient (4-15%) western blot gel (mini-protein TGX gel, Bio-Rad, Milan, Italy). Proteins 319

were transferred onto a 0.45 µm nitrocellulose membrane (Bio-Rad, Milan, Italy) at 4°C 320

for 2h at 200 mA. After transfer, the nitrocellulose membrane was blocked with 5% non-321

fat dry milk in 0.1% Tween-20 PBS (PBST) for 1h at RT. Membranes were incubated 322

overnight with rabbit anti-tagRFP (which recognise also tagBFP) or rabbit anti-323

tag(CGY)FP primary antibodies (both from Evrogen, Milan, Italy) at 1:5000 in PBST with 324

0.5% non-fat dry milk. Then, membranes were washed three times for 15 min with PBST 325

and incubated with the secondary antibody donkey anti-rabbit- IgG-HRP (sc-2317, Santa 326

Cruz Biotechnology, USA) at 1:10000 for 1h at RT. Final detection was performed using 327

enhanced chemiluminescence (ECL) Western Blotting Substrate (Amersham-Pharmacia, 328

Piscataway, NJ, USA) and image acquisition that was carried out using the ChemiDoc 329

System (Bio-Rad, Milan, Italy). Western blot analysis were repeated 4 times. 330

331

Desiccation of sheep fibroblasts expressing LEA proteins, cell viability, and residual 332

water 333

Sheep fibroblasts transfected with pTag-RAB17-GFP, pTag-WCOR410-RFP, pTag-LEA-334

BFP, individually or in combination, were detached with Trypsin-EDTA (0.25%) and 335

pelleted by spinning them for 5 min at 1200 rpm (Eppendorf Centrifuge 5804). Cells were 336


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then re-suspended to 105 cells/mL in desiccation medium (50 mM Hepes and 500 mM 337

trehalose in PBS) and drops of 10µL were placed on a plastic, cover dish. Cells were then 338

air-dried at 16°C for 1,2 3 and 4h. After desiccation, cells were rehydrated by adding 50 µL 339

of the same desiccation medium and incubated for 5 min at RT. Viability and number of 340

alive cells were evaluated using Trypan Blue staining. Un-transfected cells were used as a 341

control (CTR). Residual water was assessed by weighing first the empty cover dish, then 342

weighing the samples before drying and again after drying. This gave us a value of residual 343

water per dry weight. Desiccation and residual water assessment was done 15 times. 344

345

Cell proliferation assay 346

After desiccation, the fibroblasts were transferred into culture medium (MEM + 10% FBS) 347

and cultured in a humidified atmosphere (5% CO2/95% air at 37°C) for 24 h. Cell 348

proliferation was assessed by indirect immunocytochemistry detection of 5-bromo-2’-349

deoxyuridine (BrdU), a thymidine analogy incorporated during the S-phase of the cell 350

cycle. Briefly, cells were cultured with 100 μM BrdU for 6 hours before the end of culture, 351

fixed in cold 100% methanol for 20 min, and permeabilised with 0.1% Triton-X-100 in 352

PBS for 15 min at RT. Next, cells were treated with 4N HCl at RT for 30 min and 353

incubated with mouse anti-BrdU at 1:100 (B2531, Sigma, Milan, Italy) in blocking 354

solution (0.1% BSA in PBS) over-night at 4°C, overnight. Cells were then incubated with 355

rabbit anti-mouse IgG-FITC polyclonal antibody at 1:500 (F9137, Sigma, Milan, Italy) in 356

blocking solution at RT for 2h and counterstained with 0.5μg/mL PI at RT for 5 min. 357

Between passages, cells were washed twice with PBS at RT for 5 min. Proliferation assay 358

were repeated 5 times and at every repeat 10 different field/sample were photographed. 359

The number of proliferative cells (expressed BrdU) vs. total cells number were 360

automatically counted using Nikon Ar1 laser confocal scanning microscope (Nikon Eclipse 361

Ti-E) equipped with the NIS- Element 4.40 software. 362


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Mitochondrial and cytoskeleton staining 363

After desiccation, the fibroblasts were transferred into the culture medium (MEM + 10% 364

FBS) and cultured in a humidified atmosphere (5% CO2/95% air at 37°C) for 24h. The 365

cells were then incubated with 1 µM of Mitotracker green FM (Invitrogen, Molecular 366

Probes, Milan, Italy) for mitochondrial staining or 1 µM of Phalloidin green 367

(Thermofisher, Milan, Italy) for F-actin (the cytoskeleton) staining, both in serum-free 368

MEM for 30 min at 38.5°C. Then, cells were washed twice with PBS, fixed with 4% 369

paraformaldehyde and counterstained with 5µg/mL of Hoechst 33342. All slides were 370

examined by confocal microscopy (Nikon Eclipse Ti-E) using NIS-Elements Confocal 371

software (Nikon). 372

Statistical analysis 373

One-way ANOVA and Fisher exact test were used to compare live cells at different 374

durations of desiccation. Data reported in this paper are the mean (±SEM) for each group. 375

The level of significance was set at P < 0.05. Statistical analyses were performed using 376

GraphPad Prism for Windows (Version 6.01, GraphPad Software, Inc, CA, USA). 377

378

Acknowledgements 379

The project has received funding from the European Union’s Horizon 2020 Research and 380

Innovation Programme under the Marie Skłodowska-Curie grant agreement No 734434 381

(DRYNET) and by Narodowe Centrum Nauki (NCN) GA 2016/21/D/NZ3/02610 to MCz. 382

383

Author Contributions 384

M.C, A.F conceived and designed the research; M.C, A.F, FP.L performed the experiments; 385

M.C, A.F, FP. L, L.V analyzed the data; AP.S and PP edit the paper M.C, A.F and P.L wrote 386

the paper. All the authors discussed the results and contributed to the writing edited and 387

reviewed the manuscript. 388


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Declaration of Interests 389

The authors declare no competing interests. 390

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457

Figure legend 458

Figure 1. Subcellular localization of individual LEA proteins in sheep fibroblasts. (A-459

C) pTag-RAB17-GFP; (D – F) GFP-tag; (G - I) pTag-WCOR410-RFP; (J – L) RFP-tag; 460

(M - S) pTag-WCOR410-RFP and membrane. (A) pTag-RAB17-GFP fusion protein 461

shows cytoplasmic and nuclear localization (green arrows head, A); (B) nucleus stained 462

with Hoechst 33342; (C) merge; (D) EV control expressing only GFP was spread 463

throughout the cells; (E) nuclei stained with Hoechst 33342, (F) merge; (G) pTag-464


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WCOR410-RFP fusion protein exhibit membrane localization (red arrow); (H) nuclei 465

stained with Hoechst 33342; (I) merge; (J) RFP alone show spread localization in the cells; 466

(K) nuclei stained with Hoechst 33342 (L) merge; (M) pTag-WCOR410-RFP fusion 467

protein exhibit membrane localization (red arrow); (N) nuclei stained with Hoechst 33342; 468

(O) membrane staning, (P) merge, (R, S) enlarge Fig. 1P; (T) pTag-LEA3-BFP shows 469

mitochondrial localization, compared to (U) BFP alone that is spread throughout the cell; 470

(W – Z) pTag-LEA3-BFP co-localizes with Mitotracker green; (W) expression of pTag-471

LEA3-BFP; (X) mitochondria stained with Mitotracker green dye; (Y) nuclei stained with 472

Propidio Iode; (Z) merge; (Z’) expression of LEA proteins in sheep fibroblasts was 473

verified by western blot. 474

475

Figure 2. Subcellular localization of all three LEA proteins in sheep fibroblasts. (A) 476

pTag-WCOR410-RFP fusion protein exhibited membrane localization; (B) pTag-RAB17-477

GFP fusion protein showed cytoplasmic and nuclear localization and mitochondira stained 478

with Mitotracker green, (C) pTag-LEA-BFP showed mitochondrial localization as well as 479

nucleus stained with Hoechst 33342 (D) merge. Red - RFP; green - GFP and Mitotracker 480

green dye; blue - Hoechst 33342. 481

482

Figure 3. Enhanced resistance to desiccation stress. (A) Sheep fibroblast transfected 483

with single LEA protein or with all three LEA proteins (here called MIX). Viability of the 484

cells was controlled every hour for up to four hours using Tripan Blue staining; ** means 485

value P=0.0052 CTR vs MIX (3 and 4 hours) (B) Sheep fibroblast transfected with single 486

LEA protein or with MIX. Viability of the cells was controlled at two time points (after 1h 487

and 4h of controlled drying); **means P=0.03 CTR vs RAB17 after 1h of desiccation; *** 488

means P=0.0046 CTR vs MIX after 1h and 4h of desiccation; *p<0,05 CTR vs single 489

LEAs 4h post desiccation. 490


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491

Figure 4. LEA proteins preserve proliferation capacity following desiccation. (A) 492

Proliferation was measured by counting of cells that had incorporated BrdU in the nuclei; 493

** p<0,005; *** p<0,0005; (B) Attached cells count after desiccation, re-hydration and 494

culture for 24h in incubator [(transfected with single LEA proteins: pTag-RAB17-GFP, 495

pTag-WCOR410-RFP, pTag-LEA-BFP or all together (MIX)]; 496

497

Figure 5. Integrity of mammalian cells after desiccation. (A-H) sheep fibroblasts 498

expressing pTag-RAB17-GFP; (I-R) sheep fibroblasts expressing pTag-WCOR410-RFP; 499

(S–W) non desiccated control sheep fibroblasts (CTR); (X-Z’) non transfected sheep 500

fibroblasts, after desiccation (CTR-D); (A, O, T, Y) cytoskeleton, stained with Phalloidin 501

green; (I, N, S, X) mitochondria, stained with Mitotracker red dye; (E) mitochondria, 502

stained with Mitotracker green dye; (C, G, L, P, U, Z) nucleus, stained with Hoechst 503

33342; (D, H, M, R, W, Z’) merge; 504

505

Figure 6. pTag-LEA-BFP protects the mitochondria from desiccation damages. (A – 506

D) sheep fibroblasts expressing pTag-LEA-BFP; (E – H) control non-desiccated sheep 507

fibroblast s(CTR); (I – L) control fibroblasts subjected to desiccation (CTR-D), green - 508

mitochondria stained with Mitotracker green dye; blue - nucleus stained with Hoechst 509

33342. 510

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Figure 2.


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